Cytotoxic T cells derived from human T cell-derived iPS cells

Abstract

Provided are: cytotoxic T cells which are derived from human T cell-derived iPS cells and with which the missing-self response of NK cells can be avoided while maintaining the potent antitumor effect of an antigen-specific CTL, and allogeneic administration is possible; and a method for producing the same.　This method, which is for producing cytotoxic T cells derived from human T cell-derived iPS cells and by which HLA restriction class I of an antigen epitope of CTL and HLA class I of HLA-E are expressed, is characterized by comprising: a step for knocking out all antigens in the HLA class I of human T cell-derived iPS cells; a step for introducing genes of the HLA-E and HLA restriction HLA class I of an antigen epitope of CTL into T-iPS cells, in which all the antigens in the HLA class I have been knocked out; and a step for redifferentiating the gene-introduced T-iPS cells into CD8 single positive T cells.

Description

[Technical Field]

[0001] The present invention relates to cytotoxic T cells derived from human T cell-derived iPS cells that can be administered to other animals, and a method for producing the same. [Background technology]

[0002] Antigen-specific cytotoxic T cells (CTLs) recognize antigenic peptides derived from viruses, tumors, etc., presented together with class 1 major histocompatibility complexes (MHC class I, HLA class I) on antigen-presenting cells via T cell receptors (TCRs) present on their cell surfaces, and exert specific cytotoxic activity against foreign cells presenting the antigenic peptides. Furthermore, some CTLs become long-lived memory T cells, retaining their cytotoxicity against foreign substances and remaining in the host's memory, allowing them to respond to subsequent exposure to foreign substances. Therefore, CTLs are expected to be used as immune cell therapy for patients with viral infections and cancer.

[0003] In patients with chronic viral infections or cancer, chronic exposure to antigens causes T cells to become exhausted and senescent, rendering them ineffective, and therefore ineffective in many cases. Therefore, iPS cell-derived rejuvenated T cell therapy, in which exhausted T cells are functionally rejuvenated using iPS cell technology and the resulting rejuvenated T cells are administered to patients, is expected to be an effective means of improving the efficacy of cancer treatment. To obtain CTLs for this rejuvenated T cell therapy, a method has been developed in which T cell-derived iPS cells (T-iPS cells) are established from antigen-specific T cells, and then the cells are redifferentiated into CTLs, chimeric antigen receptor T cells (CART), etc., while maintaining the recombinant structure of the TCR gene of the original T cell (Patent Document 1).

[0004] However, these methods have the drawback of taking five months to produce them and being expensive. In contrast, if allogeneic CTL-derived iPS cells are stockpiled in advance and rejuvenated T cells (rejT) are produced, they can be administered to patients more quickly and the cost per patient can be reduced. However, if the HLA does not match, the cells will be rejected, resulting in a weakened anti-tumor effect.

[0005] To address this issue, eliminating HLA class I expression and generating allogeneic rejT cells that are not rejected by patient CD8+ T cells could enable rapid administration to many severely ill patients while maintaining the potent antitumor activity of antigen-specific CTLs. To enable the use of iPS cells derived from healthy donors and validation stockpiles, HLA genome editing to knock out B2M and eliminate HLA class I antigens is a promising approach. However, this approach requires suppressing the missing-self response of NK cells. Several editing methods have been reported to avoid the missing-self response of NK cells, including eliminating HLA class I in iPS cells to express only HLA-E, thereby inducing differentiation and avoiding NK cell attack (Non-Patent Document 1), eliminating HLA class I and class II and then expressing PD-L1, HLA-G, and the "don't-eat-me" signal CD47 (Non-Patent Document 2), and eliminating HLA-A and -B while retaining HLA-C (Non-Patent Document 3). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6164746 [Non-patent literature]

[0007] [Non-Patent Document 1] Gornalusse GG, Hirata RK, Funk SE, Riolobos L, Lopes VS, Manske G, et al. HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nature biotechnology. 2017;35(8):765-72. [Non-patent document 2] Han X, Wang M, Duan S, Franco PJ, Kenty JH, Hedrick P, et al. Generation of hypoimmunogenic human pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America. 2019;116(21):10441-6. [Non-patent document 3] Xu H, Wang B, Ono M, Kagita A, Fujii K, Sasakawa N, et al. Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility. Cell stem cell. 2019;24(4):566-78 e7. Summary of the Invention [Problem to be solved by the invention]

[0008] However, none of these methods was sufficiently satisfactory in terms of the balance between avoiding the missing-self response of NK cells and maintaining the antigen-specific CTL activity that they inherently possess. Therefore, an object of the present invention is to provide cytotoxic T cells derived from human T cell-derived iPS cells that can be administered allogeneically and that can avoid the missing-self response of NK cells while maintaining the potent antitumor effect of antigen-specific CTLs, and a method for producing the same. [Means for solving the problem]

[0009] Therefore, the present inventors discovered that the activity of NK cells can be more potently suppressed by expressing HLA-restricted class I molecules of CTL antigen epitopes (e.g., HLA-A24 for HLA-A24-restricted CTLs, and HLA-A02 for HLA-A02-restricted CTLs) and HLA-E in iPS cells derived from human T cells, and thus completed the present invention.

[0010] That is, the present invention provides the following [1] to [4]. [1] Cytotoxic T cells derived from human T cell-derived iPS cells that express HLA-restricted class I molecules of CTL antigen epitopes and HLA class I of HLA-E. [2] Cytotoxic T cells derived from human T cell-derived iPS cells according to [1], wherein the HLA-restricted class I molecule of the antigen epitope of the CTL is HLA-A24 or HLA-A02. [3] A method for producing cytotoxic T cells derived from human T cell-derived iPS cells that express two types of HLA class I, namely, HLA-restricted class I molecules of CTL antigen epitopes and HLA-E, comprising the steps of: knocking out all HLA class I in human T cell-derived iPS cells; introducing genes for HLA-restricted class I molecules of CTL antigen epitopes and HLA-E into the T cells from which all HLA class I have been knocked out; and redifferentiating the gene-introduced T-iPS cells into CD8 single-positive T cells. [4] The method according to [3], wherein the HLA-restricted class I molecule of the CTL antigen epitope is HLA-A24 or HLA-A02. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide cytotoxic T cells derived from human T cell-derived iPS cells that can be administered allogeneically, and which can avoid the missing-self response of NK cells while maintaining the potent antitumor effect of antigen-specific CTLs, as well as a stable method for producing the same. [Brief explanation of the drawings]

[0012] [Figure 1] This figure shows HLA class I expression in HPV-rejT after genome editing. Post-edit HPV-rejT shows HPV-rejT in which only HLA-A24 was knocked in after HLA class I loss (top row), and HPV-rejT expressing both HLA-A24 and HLA-E (bottom row). HPV-rejT was stained with antibodies against each HLA class I (ABC, A24, BC, E) and analyzed by flow cytometry. Control (isotype) shows a negative control in which HPV-rejT was stained with an isotype control. [Figure 2] This figure shows that HPV-rejT expressing both HLA-A24 and HLA-E significantly suppresses the cytotoxic activity of NK cells. The K562 cell line is a positive control that does not express class I and is therefore cytosed by NK cells. KI-A24 indicates HPV-rejT in which only HLA-A24 was knocked in after HLA class I loss. KI-E indicates HPV-rejT in which only HLA-E was knocked in after HLA class I loss. KI-A24&E indicates HPV-rejT in which both HLA-A24 and HLA-E were knocked in after HLA class I loss. WT indicates wild-type HLA-expressing cells without HLA editing. Auto CTL indicates CTL derived from the donor of the NK cells used in this analysis. Because auto CTLs are autologous, NK cells do not attack auto CTLs, so they were used as a negative control. [Figure 3]This figure shows that HLA-A24+HLA-E expressing HPV-rejT strongly suppresses NK cytotoxicity in the 107a assay. Because the K562 cell line does not express class I, NK cells attack K562 cells in co-culture, resulting in high expression of 107a. This was used as a positive control. Positive Ctrl indicates NK cells stimulated with Cell Stimulation Cocktail, resulting in high expression of 107a. This also indicates the positive control. HLA-E KI indicates HPV-rejT in which only HLA-E was knocked in after HLA class I loss. HLA-A24 KI indicates HPV-rejT in which only HLA-A24 was knocked in after HLA class I loss. HL-A24+E KI indicates HPV-rejT in which both HLA-A24 and HLA-E were knocked in after HLA class I loss. Negative Ctrl indicates a negative control cultured with NK cells alone. [Figure 4] 1 shows the survival time extension effect of HLA-edited HPV-rejT on cervical cancer-bearing mice. no treatment indicates the non-treatment group, original CTL indicates the original CTL-treated group, WTrejT indicates the rejT-treated group, and EXrejT indicates the HPV-rejT-treated group of the present invention. [Figure 5] This shows the in vivo durability of HLA-edited HPV-rejT when co-administered with NK cells. The number in parentheses in HPV-rejT indicates HLA class I editing. DETAILED DESCRIPTION OF THE INVENTION

[0013] The cytotoxic T cells of the present invention are cytotoxic T cells in which the HLA of human T cell-derived iPS cells (T-iPS cells) has been modified to express HLA-restricted class I molecules of CTL antigen epitopes and HLA class I of HLA-E. Furthermore, the cytotoxic T cells of the present invention may express HLA class I molecules such as HLA-G and HLA-C in addition to HLA-E and HLA-restricted class I molecules of the CTL antigen epitope, and may also express CD47, PD-L1, iCaspase 9, etc. However, considering the suppression of NK cell activity and the need to match the HLA with that of the donor due to HLA polymorphism, cytotoxic T cells derived from human T cell-derived iPS cells are preferred, expressing two types of HLA class I molecules, HLA-E and HLA-restricted class I molecules of the CTL antigen epitope.

[0014] The human T cell-derived iPS cells used as a starting material can be obtained by inducing human T cells into iPS cells (T-iPS cells). The method for producing these T-iPS cells is preferably carried out by the method described in Patent Document 1.

[0015] First, we will explain how to induce iPS cells from human T cells. The T cells used are preferably human T cells. The human source of these T cells may be a human suffering from a viral infection, malignant tumor, or the like, but is preferably a healthy individual, in order to produce therapeutic alloantigen-specific cytotoxic T cells, which can be banked after genome editing and administered to many people. Furthermore, the preferred human source of T cells does not need to have an HLA type that is completely identical to that of the patient to whom regenerated CTLs or CART cells produced using T-iPS cells produced by the present invention will be administered.

[0016] In the present invention, the T cells induced to become T-iPS cells are preferably T cells with antigen specificity. Examples include T cells expressing CD3 and CD8, specifically CD8-positive cells (CTLs). Other examples include T cells expressing CD3 and CD4, specifically CD4-positive cells. The antigen specificity of T cells is conferred by antigen-specific rearranged TCR genes. From the viewpoint of production efficiency, although not limited thereto, antigen-specific CD8-positive T cells are preferably used as the human T cells induced to become T-iPS cells to obtain antigen-specific CD8-positive cells, and antigen-specific CD4-positive T cells are preferably used as the human T cells induced to become T-iPS cells to obtain antigen-specific CD4-positive cells. Furthermore, when performing immunotherapy, it is preferable that the human T cells differentiated from iPS cells have the same or substantially the same antigen specificity as the human T cells induced to become iPS cells. Furthermore, T cells that induce T-iPS cells also include T cells without antigen specificity. Specifically, examples include genetically modified T cells such as CART cells or TCR-T cells.

[0017] Such T cells can be isolated, for example, from human tissue by known techniques. Human tissues include tissues containing T cells, such as peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and lesion tissue. Among these, peripheral blood is preferred because it is less invasive to humans and easier to prepare. Tumor-infiltrating lymphocytes (TILs) can be isolated from tumor tissue or peripheral blood. Known techniques for isolating human T cells include magnetic selection using magnetic beads for cell separation, flow cytometry using a cell sorter and antibodies against cell surface markers such as CD4 or CD8, and activated T cell induction methods using anti-CD3 and anti-CD28 antibodies. Desired T cells can also be isolated using cytokine secretion, functional molecule expression, or signal molecules such as PD-1 as indicators. Cytotoxic T cells (CTLs) can also be isolated using secretion or production of granzymes or perforins as indicators. Furthermore, when isolating T cells having antigen specificity from human tissues containing such cells, T cells having the desired antigen specificity can be purified from human tissues using a multimer of MHC (major histocompatibility complex) bound to the desired antigen (e.g., "MHC tetramer" or "Pro5 (registered trademark) MHC class I pentamer").

[0018] In the present invention, the genes introduced to convert T cells into iPS cells are preferably a combination of at least four of the following genes: (a) Oct3 / 4 gene, (b) c-Myc gene, (c) Sox2 gene, (d) Klf4 gene, (e) NANOG gene, and (f) LIN28 gene.

[0019] In the present invention, the method for introducing the gene group into T cells is not particularly limited, and any known method can be appropriately selected and used. For example, when the gene group is introduced into T cells in the form of nucleic acids encoding the gene group, the nucleic acid (e.g., cDNA, RNA) encoding the gene group can be inserted into an appropriate expression vector containing a promoter that functions in T cells, and the expression vector can be introduced into cells by infection, lipofection, liposome method, electroporation, calcium phosphate co-precipitation, DEAE-dextran method, microinjection, or electroporation.

[0020] Among these expression vectors, it is more preferable to use a stealth RNA expression vector containing the above-mentioned gene group, in terms of reducing the risk of canceration and introducing efficiency. Stealth RNA expression vectors are designed to avoid chromosomal incorporation and to achieve sustained and stable gene expression in the cytoplasm rather than the nucleus. They can be used to introduce large genes of 13,000 base pairs or more, or even 10 genes at a time, without harming cells. They can be removed when the introduced gene is no longer needed, and their stealth properties mean that cells cannot recognize the vector as a foreign body. Such stealth RNA expression vectors include a complex that does not activate innate immune structures and consists of a minus single-stranded RNA (A) containing the RNA sequences (1) to (8) below, a single-stranded RNA-binding protein (B), and an RNA-dependent RNA polymerase. (1) RNA sequences for the gene group; (2) a human mRNA-derived RNA sequence that constitutes a non-coding region; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA synthetase; (4) a transcription termination signal sequence recognized by the RNA-dependent RNA synthetase; (5) an RNA sequence containing a replication origin recognized by the RNA-dependent RNA synthetase; (6) an RNA sequence encoding the RNA-dependent RNA synthetase; (7) an RNA sequence encoding a protein that regulates the activity of the RNA-dependent RNA synthetase; (8) An RNA sequence encoding the single-stranded RNA-binding protein.

[0021] Furthermore, when establishing T-iPS cells, the T cells are preferably activated by stimulation with anti-CD3 and anti-CD28 antibodies in the presence of interleukin-2 (IL-2) or interleukin-7 (IL-7) and interleukin-15 (IL-15) before the introduction of the gene cluster. Alternatively, the T cells may be stimulated with at least one substance selected from the group consisting of phytohemagglutinin (PHA), interleukin-2 (IL-2), alloantigen-expressing cells, anti-CD3 and anti-CD28 antibodies, and CD3 and CD28 agonists. Such stimulation can be carried out, for example, by adding PHA, IL-2, anti-CD3 and / or anti-CD28 antibodies to a medium and culturing the T cells for a certain period of time. Furthermore, the anti-CD3 and anti-CD28 antibodies may be bound to magnetic beads or the like. Furthermore, instead of adding these antibodies to the medium, the T cells may be stimulated by culturing them for a certain period of time on a culture dish to which anti-CD3 and anti-CD28 antibodies are bound. Furthermore, the T cells (for example, human T cells) may be stimulated by adding an antigen peptide recognized by the T cells to the medium together with the feeder cells.

[0022] To provide such stimulation to the T cells, the concentration of PHA added to the medium is not particularly limited, but is preferably 1 to 100 μg / mL.Furthermore, the concentration of IL-2 added to the medium is not particularly limited, but is preferably 1 to 200 ng / mL. Furthermore, the concentrations of anti-CD3 antibody and anti-CD28 antibody added to the medium are not particularly limited, but are preferably 1 to 10 times the culture volume of the T cells. Furthermore, the concentrations of anti-CD3 antibody and anti-CD28 antibody bound to the surface of the culture dish to provide such stimulation to the T cells are not particularly limited, but the concentrations upon coating are preferably 0.1 to 100 μg / mL, preferably 1 to 100 μg / mL, for anti-CD3 antibody and 0.1 to 10 μg / mL for anti-CD28 antibody.

[0023] The culture period for such stimulation is not particularly limited, as long as it is a period sufficient to provide such stimulation to the T cells and to allow the T cells to proliferate to the number of cells required for the introduction of the four genes, but is usually 2 to 7 days, and from the viewpoint of gene transfer efficiency, is preferably 3 to 5 days. Infection is preferably carried out by mixing the T cells with the vector in a 15 mL tube, or, from the viewpoint of increasing gene transfer efficiency, culture is preferably carried out on a culture dish coated with Retronectin.

[0024] Examples of the medium to which the T cells are cultured and to which PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody, etc. are added include known media suitable for culturing the T cells (more specifically, Roswell Park Memorial Institute (RPMI) 1640 medium, AIM V medium, etc., which contain other cytokines and human serum). TM The medium may contain, in addition to PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody, amino acids (e.g., L-glutamine) and antibiotics (e.g., streptomycin and penicillin) necessary for culture. It is also preferable to add IL-7 and IL-15 to the medium instead of IL-2. The concentrations of IL-7 and IL-15 added are not particularly limited, but are preferably 1 to 100 ng / mL each.

[0025] Furthermore, there are no particular limitations on the conditions for or after introducing the four genes into the T cells. However, it is preferable to culture the T cells into which the four genes have been introduced under feeder-free conditions. Examples include wells coated with iMatrix-511 solution, which is a laminin 511E8 fragment, or vitronectin. Cultures can also be established under feeder cell conditions. Examples of feeder cells include mouse embryonic fibroblasts (MEF), STO cells, and SNL cells whose cell division has been arrested by irradiation or antibiotic treatment.

[0026] Furthermore, during the process of inducing T cells to T-iPS cells, it is preferable to add iPS cell medium from the next day, and then replace the medium by half every other day, gradually replacing the T cell medium with iPS medium.

[0027] Furthermore, it is preferable to culture the iPS cells while gradually replacing a known medium suitable for culturing the T cells with a medium suitable for culturing iPS cells as the T cells transition from the iPS cells to the iPS cells. A known medium can be appropriately selected and used as the medium suitable for culturing iPS cells. For example, StemFit AK03N is preferable for iMatrix-coated iPS cells, or Essential 8 Medium is preferable for vitronectin-coated iPS cells. For feeder cells such as MEF cells, Dulbecco's modified Eagle's medium / F12 medium (human iPS cell culture medium) containing knockout serum substitute, L-glutamine, non-essential amino acids, 2-mercaptoethanol, b-FGF, and the like is preferable.

[0028] In this way, T-iPS cells can be selected by appropriately selecting known techniques. Examples of such known techniques include selection by observing the morphology of ES cell / iPS cell-like colonies under a microscope. On the other hand, in the case of T-iPS cells established from single-cell CTL clones, their properties are often similar, so an alternative method is to simply passage all established colonies without selecting each T-iPS cell colony.

[0029] The identity of the cells selected in this manner as T-iPS cells can be confirmed by, for example, detecting the expression of undifferentiated cell-specific markers (ALP, SSEA-4, Tra-1-60, Tra-1-81, etc.) in the selected cells by immunostaining, RT-PCR, or by transplanting the selected cells into mice and observing the formation of teratomas. Furthermore, the identity of the cells selected in this manner as T cells can be confirmed by detecting the state of TCR gene rearrangement by genomic PCR.

[0030] The time to select and recover these cells is preferably 10 to 40 days, preferably 14 to 28 days, after the gene group containing the four genes is introduced into the T cells. Unless otherwise specified, the culture environment is preferably 5% CO2, 35 to 38°C, more preferably 5% CO2, 37°C.

[0031] To generate T cells that express HLA-restricted class I genes of CTL antigen epitopes and HLA class I of HLA-E from the human T cell-derived iPS cells obtained as described above, for example, a method comprising the steps of (a) knocking out all HLA class I genes of human T cell-derived iPS cells, and (b) introducing HLA-restricted class I genes of CTL antigen epitopes and HLA-E genes into T-iPS cells from which all HLA class I genes have been knocked out.

[0032] The above knockout step (a) and gene introduction step (b) can be carried out by various methods, but the CRISPR-Cas9 genome editing method is one option. To knock out all HLA class I genes in human T cell-derived iPS cells using CRISPR-Cas9 genome editing, first knock out β2-microglobin (B2M). After cell detachment, 5 μg each of the knockout plasmid and guide RNA was added to 2 × 10 5Approximately 10 T-iPSCs are electroporated. They are then seeded into three wells of a six-well plate and cultured. The knockout plasmid contains the target sequence of the guide RNA used for the second editing, as well as GFP and selection markers such as CD8 and CD19. Approximately 10 days later, when the cells seeded into the three wells become confluent, positive selection of the selection marker is performed. After selection, the iPS cells are single-cell cloned by thinly seeding the T-iPSCs. Strongly GFP-positive cells are picked, cultured, and genotyped. Clones containing the marker in the biallelic region are identified by PCR and expanded before proceeding to the next step.

[0033] Next, in step (b), HLA-restricted class I genes and HLA-E genes are introduced into T-iPSCs in which all HLA class I genes have been knocked out using CRISPR-Cas9 genome editing. T-iPSCs in which B2M was knocked out in the first edit are detached and electroporated with 2.5 μg each of two knock-in plasmids and 5 μg of guide RNA. After electroporation, the cells are seeded into three wells of a 6-well plate and cultured. Approximately seven days later, when the cells seeded into the three wells become confluent, negative selection of the selection marker is performed. After selection, the iPS cells are thinly seeded to perform single-cell cloning. GFP-negative cells are picked, cultured, and genotyped. Clones containing the marker in the biallelic region are identified by PCR and expanded. Furthermore, by using the same methods as described above, in addition to the HLA-restricted class I molecules of the CTL antigen epitope and HLA-E, HLA class I molecules such as HLA-G and HLA-C may be expressed, and CD47, PD-L1, iCaspase9, etc. may also be expressed.

[0034] Next, the genome-edited T-iPS cells are induced to differentiate into CTL cells. The preferred method for inducing redifferentiation is to differentiate T-iPS cells into CD8+ single-positive T cells, and more preferably to differentiate T-iPS cells into CD4 / CD8 double-negative T cells and then differentiate the CD4 / CD8 double-negative T cells into CD8+ single-positive T cells. Furthermore, as described in Patent Document 1, it is preferable to obtain these cells by differentiating T-iPS cells into CD4 / CD8 double-negative cells, stimulating the CD4 / CD8 double-negative cells by adding a substance that stimulates the T cell receptor, and then differentiating the CD4 / CD8 double-negative cells whose T cell receptors have been stimulated into CD8 single-positive T cells in the presence of the cytokines IL-7 and IL-15.

[0035] To differentiate T-iPS cells into CD4 / CD8 double-negative cells, it is preferable to culture T-iPS cells on feeder cells (preferably mouse stromal cells) in a medium containing cytokines, serum (e.g., fetal bovine serum (FBS)), insulin, transferrin, sodium selenite, L-glutamine, α-monothioglycerol, ascorbic acid, etc. The stromal cells used are preferably OP9 cells or 10T1 / 2 cells (C3H10T1 / 2 cells) that have been treated with irradiation or other methods. The cytokine added to the medium is preferably at least one cytokine selected from the group consisting of VEGF, SCF, TPO, SCF, and FLT3L, and more preferably VEGF, SCF, and TPO, or VEGF, SCF, and FLT3L. Examples of media include X-VIVO medium, Iscove's Modified Dulbecco's Medium (IMDM medium), α-MEM, and DMEM. IMDM medium is preferred because it facilitates the formation of T-iPS sacs (sac-like structures containing hematopoietic progenitor cells). The culture period for these T-iPS cells is preferably 8 to 14 days, more preferably 10 to 14 days, from the initiation of T-iPS cell culture. The culture environment is not particularly limited, but is preferably 5% CO2 at 35 to 38°C, more preferably 5% CO2 at 37°C. Furthermore, it is more preferable to culture the cells under low oxygen concentration conditions (oxygen concentration: for example, 5 to 20%) for about a week.

[0036] To differentiate T-iPS cells into CD4 / CD8 double-negative cells, the cells contained in the T-iPS sac are preferably cultured on feeder cells (preferably stromal cells, more preferably human stromal cells) in a medium containing cytokines, serum (e.g., FBS), and other factors. Cells present in the T-iPS sac can be separated, for example, by passing them through a sterilized sieve (e.g., a cell strainer). The stromal cells used for this culture are preferably OP9-DL1 cells, OP9-DL4 cells, 10T1 / 2 / DL4 cells, or 10T1 / 2 / DL1 cells that have been treated with irradiation or other methods, in order to induce differentiation into T lymphocytes via Notch signaling. Examples of cytokines added to the medium include IL-7, FLT3L, VEGF, SCF, TPO, IL-2, and IL-15. Examples of media include α-MEM medium, DMEM medium, and IMDM medium, with α-MEM medium being preferred. In addition to IL-7 and FLT3L, the medium may also contain amino acids (eg, L-glutamine) and antibiotics (eg, streptomycin and penicillin) necessary for culture.

[0037] The culture period for the cells contained in the T-iPS sac is preferably the period until the CD4 / CD8 double-negative cells differentiated in this manner begin to express T cell receptors (TCRs) on their surface, and is preferably 14 to 28 days from the start of culture of the cells contained in the T-iPS sac. The culture environment is not particularly limited, but is preferably 5% CO2, 35 to 38°C, and more preferably 5% CO2, 37°C.

[0038] Whether or not T cell receptors (TCRs) are expressed on the cell surface of CD4 / CD8 double-negative cells can be evaluated by flow cytometry using anti-TCRαβ antibodies, anti-CD3 antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.

[0039] In the method for producing antigen-specific human CD8 single-positive cells, further rearrangement of the TCR gene can be suppressed by stimulating T-iPS cell-derived CD4 / CD8 double-negative cells via the TCR expressed on the cell surface, thereby extremely increasing the frequency of T cells with the same TCR gene rearrangement pattern as the original human T cell among the CD8 single-positive cells obtained by redifferentiation.

[0040] A preferred method for stimulating the T cell receptors of T-iPS cell-derived CD4 / CD8 double-negative cells is to contact the T-iPS cell-derived CD4 / CD8 double-negative cells with at least one substance selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, an antigenic peptide that specifically binds to the human T cells from which the T-iPS cells were derived, cells expressing an HLA complex that restricts the T cell receptor, and an MHC multimer to which the antigenic peptide is bound.From the perspective of providing physiological stimulation, contact with cells expressing a specific peptide / HLA complex is more preferred.Furthermore, from the perspective of emphasizing uniformity of stimulation, contact with an antibody or reagent is more preferred.

[0041] The contact can be carried out, for example, by adding PHA or the like to the medium and culturing the T cells for a certain period of time. The anti-CD3 antibody and anti-CD28 antibody may be bound to magnetic beads or the like. Instead of adding these antibodies to the medium, the T cells may be stimulated by culturing them for a certain period of time on a culture dish with anti-CD3 antibody and anti-CD28 antibody bound to its surface. Furthermore, stimulation may also be achieved by adding the antigen peptide to the medium together with feeder cells.

[0042] To stimulate the TCR of CD4 / CD8 double-negative cells, the concentration of PHA added to the medium is preferably 1 to 100 μg / ml. Furthermore, the concentrations of anti-CD3 antibody and anti-CD28 antibody added to the medium are preferably 1 to 10 times the culture volume of the T cells. Furthermore, to stimulate the TCR of CD4 / CD8 double-negative cells, the concentrations of anti-CD3 antibody and anti-CD28 antibody bound to the surface of a culture dish at the time of coating are preferably 0.1 to 100 μg / ml for anti-CD3 antibody and 0.1 to 10 μg / ml for anti-CD28 antibody.

[0043] The culture period for the cells contained in the T-iPS sac preferably includes the period required for the expression of T cell receptors (TCRs) on the cell surface of the CD4 / CD8 double-negative cells obtained by differentiation in this manner, and is preferably 7 to 29 days from the start of culture of the cells contained in the T-iPS sac. The culture environment is preferably 5% CO2, 35 to 38°C, more preferably 5% CO2, 37°C.

[0044] In the present invention, in order to differentiate CD4 / CD8 double-negative cells whose T cell receptors have been stimulated into CD8 single-positive cells, the CD4 / CD8 double-negative cells are preferably cultured in a medium containing cytokines, serum (e.g., human serum), and the like. The cytokines added to the medium may be any cytokine capable of differentiating CD4 / CD8 double-negative cells into CD8 single-positive cells, such as IL-7 and IL-15. Among these, adding IL-7 and IL-15 in combination is preferred, from the viewpoint of selecting the CD8 lineage and facilitating the generation of memory CD8 T cells during differentiation into CD8 single-positive cells. The concentrations of IL-7 and IL-15 added are preferably 1 to 20 ng / ml. Examples of media include RPMI-1640 medium, X-VIVO medium, DMEM medium, and α-MEM medium, with RPMI-1640 medium or X-VIVO medium being preferred. In addition to IL-7, IL-15, etc., the medium may also contain amino acids necessary for culture (e.g., L-glutamine), antibiotics (e.g., streptomycin, penicillin), and cytokines other than IL-7, IL-15, etc.

[0045] In such culture, the CD4 / CD8 double-negative cells may be co-cultured with feeder cells. The feeder cells are preferably peripheral blood mononuclear cells (PBMCs). These PBMCs are preferably allogeneic to the CD4 / CD8 double-negative cells. From the viewpoint of continuously stimulating TCR and continuously suppressing further TCR rearrangement, it is more preferable to use peripheral blood mononuclear cells that present antigen peptides that specifically bind to the human T cells that are the source of the CD4 / CD8 double-negative cells.

[0046] The culture period for differentiating these CD4 / CD8 double-negative cells into CD8 single-positive cells is preferably 2 to 4 weeks, and the culture environment is preferably 5% CO2, 35 to 38°C, more preferably 5% CO2, 37°C.

[0047] The CD8 single-positive cells induced to differentiate in this manner can be confirmed to be derived from T-iPS cells and the T cells from which the T-iPS cells were derived, for example, by detecting the state of TCR gene rearrangement by genomic PCR.

[0048] Furthermore, the CD8 single-positive cells thus obtained can be isolated by appropriately selecting a known technique. Examples of such known techniques include flow cytometry using an antibody against the CD8 cell surface marker and a cell sorter. For example, in the case of CD8 single-positive cells, a purification method using an affinity column immobilized with an antigen recognized by the T cells from which the CD8 single-positive cells were derived, or a purification method using an MHC multimer (e.g., MHC tetramer) bound to the antigen can also be employed.

[0049] Furthermore, the CD8 single-positive cells obtained according to the present invention do not express PD-1, but express CCR7 along with CD27 and CD28, which are representative of the central memory T cell phenotype; their telomeres are also longer than those of the original T cells, and they have high self-renewal ability. Therefore, according to the present invention, it is possible to produce CD8 single-positive T cells that have the same TCR gene rearrangement pattern as the original T cells, but which do not express PD-1 and express CD27, CD28, and CCR7. Furthermore, T cells collected from humans differ from the obtained T cells in that they express PD-1 and have a low proportion of the immature memory phenotype.

[0050] To maintain the CD8 single-positive cells obtained in this manner, the cells may be stimulated every 1 to 2 weeks. Such stimulation may involve contact with at least one substance selected from the group consisting of anti-CD3 antibody, anti-CD28 antibody, IL-2, IL-7, IL-15, an antigen recognized by the CD8SP cells, an MHC multimer to which the antigen is bound, feeder cells in an allogeneic relationship with the CD8 single-positive cells, and feeder cells in an autogeneic relationship with the CD8 single-positive cells.

[0051] The thus obtained cytotoxic T cells derived from human T cell-derived iPS cells of the present invention, which express HLA class I HLA-A24 and HLA-E, are capable of avoiding the missing-self response of NK cells while maintaining the antigen-specific cytotoxicity of the human T cells used as the starting material, and are therefore useful as cytotoxic T cells derived from human T cell-derived iPS cells that can be administered to allogeneic recipients. The missing-self response of NK cells from the cytotoxic T cells of the present invention is significantly lower than that of T cells expressing only HLA-A24 or HLA-E. [Example]

[0052] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0053] Example 1 Establishment of T-iPS cells from human papillomavirus (HPV)-specific CTL clones using Sendai virus vectors. 1) Peripheral blood mononuclear cells were isolated from the peripheral blood of healthy donors, and dendritic cells were induced for antigen presentation. Seven days later, HPV antigen peptides (HPV16-E6, A2402) were added to the induced dendritic cells, and co-culture with peripheral blood mononuclear cells was initiated. Approximately 8-10 days later, to detect HPV-specific CTLs, CTLs were stained with MHC tetramers and the tetramer positivity rate was confirmed by flow cytometry. After HPV-specific CTLs were identified, single-cell sorting or tetramer / PE bead selection was performed, followed by limiting dilution. Then, PBMCs irradiated with 50 Gy of X-rays were stimulated with IL2 and PHA. 2) After approximately 3-6 weeks, colonies that had formed were stained with tetramers, and the establishment of CTL clones was confirmed by flow cytometry. After confirming establishment, the CTL clones were stimulated with CD3 / 28 and then transfected with the two vectors listed in A). The transfected CTLs were transferred to a 6-well plate coated with iMatrix, and culture was initiated in a CO2 incubator using CTL medium.

[0054] A) SeV4 vector + SV40 large T antigen

[0055] 3) The day after SeV gene transfection, an equal volume of iPS medium (StemFitAK03N) was added, and thereafter half the volume was replaced with StemFitAK03N every other day. 4) After 7 days, colonies of T-iPS cells were observed, and colonies were then picked.

[0056] Example 2 Knockout of all HLA class I receptors in iPS cells derived from HPV antigen-specific CTLs. First, β2-microglobulin (B2M) was knocked out. After cell detachment, T-iPSCs were electroporated with 5 μg each of the knockout plasmid and guide RNA using a LONZA 4-D Nucleofector. The cells were then seeded into three wells of a six-well plate and cultured. The knockout plasmid contained the target sequence of the guide RNA used for the second editing, as well as GFP and CD8 selection markers. Approximately 10 days later, when the cells seeded into the three wells reached confluence, MACS bead positive selection for CD8 was performed. After selection, T-iPSCs were thinly seeded for single-cell cloning of iPSCs. Strongly GFP-positive cells were picked, cultured, and genotyped. Clones containing biallelic markers were identified by PCR and expanded for the next step.

[0057] Example 3 Introduction of HLA-A24 and HLA-E genes. Because the epitope is A2402-restricted, HLA-A2402 was knocked into T-iPSCs after B2M knockout. Simultaneously, a knock-in plasmid with an HLA-E trimer structure was created to knock in HLA-E. Furthermore, a plasmid was created in which half of HLA-A2402 and half of HLA-E were knocked in simultaneously. T-iPSCs with B2M knocked out in the first edit were detached and electroporated using a LONZA 4-D Nucleofector. After electroporation, the cells were seeded into three wells of a 6-well plate and cultured. Approximately seven days later, when the cells seeded in the three wells reached confluence, negative CD8 selection was performed using MACS beads. After selection, T-iPSCs were thinly seeded to allow single-cell cloning of iPSCs. GFP-negative cells were picked and cultured, genotyped, and clones were identified and expanded.

[0058] Example 4 Redifferentiation of genome-edited T-iPS cells into CD8 single-positive T cells. Small clusters of genome-edited T-iPS cells obtained in Example 3 were transferred onto C3H10T1 / 2 cells and co-cultured in EB medium in the presence of 20 ng / mL VEGF, 50 ng / mL SCF, and 50 ng / mL FLT-3L. On day 14 of culture, hematopoietic cells contained in the iPS sacs were collected and transferred onto DL1 / 4-expressing C3H10T1 / 2 cells. The hematopoietic cells were differentiated into T lineage cells in OP9 medium in the presence of 10 ng / mL FLT-3L and 1 ng / mL IL-7.

[0059] Then, on day 42 of the culture, α-CD3 / CD28 beads or 5 μg / ml PHA were added to the OP9 medium for stimulation.

[0060] The T lineage cells were then collected and cultured in CTL medium together with irradiated PMBCs in the presence of 10 ng / mL IL-7 and 10 ng / mL IL-15.

[0061] On day 56 of culture, CD8 / tetramer-positive cells were observed. FACS analysis of these cells revealed that the knocked-in HLA genes (HLA-A24, HLA-E, or both) were expressed.

[0062] Example 5 Antigen specificity of the CD8 single positive cells of the present invention. We investigated whether the redifferentiated cells obtained in Example 4 retained the same antigen specificity as the original T cells after genome editing. The results showed that the redifferentiated CD8 single-positive cells obtained had the same antigen specificity as the original HPV-T cell clone (Figure 1). After successfully inducing differentiation of rejuvenated CTLs (rejT) expressing only HLA-A24, only HLA-E, or both, we performed chromium assays and CD107a assays to examine whether they could prevent NK cell missing-self responses. HPV-rejT expressing only HLA-A24 or only HLA-E suppressed NK activity, but not sufficiently, whereas HPV-rejT expressing both HLA-A24 and HLA-E significantly suppressed NK cell cytotoxicity. These results demonstrate that the expression of both HLA-restricted HLA class I molecules and HLA-E, which are antigen epitopes of CTLs, is important for suppressing NK activity (Figures 2 and 3).

[0063] Example 6 The survival benefit of HLA-edited HPV-rejT in cervical cancer-bearing mice. (method) Immunodeficient mice (NOG mice) were intraperitoneally transplanted with the cervical cancer cell line SiHa. They were divided into an untreated control group and three treatment groups (the original HPV-CTL clone, wild type (WT) HPV-rejT, or HLA-edited HPV-rejT) and received 2.5x10 6 T cells were administered intraperitoneally three times. The survival time of mice in each treatment group was compared with that of the untreated control group to confirm the therapeutic effect. (result) The survival time of cervical cancer-bearing mice is shown in FIG. As shown in Figure 4, the survival period of cervical cancer-bearing mice was significantly extended in the WT rejT administration group and the HLA-edited HPV-rejT (EXrejT) administration group compared to the original CTL administration group. Furthermore, pathological examination of treated mice that survived for more than six months revealed no residual tumors in the lungs, liver, intestines, spleen, or uterus.

[0064] Example 7 In vivo durability of HLA-edited HPV-rejT upon coadministration with NK cells. (method) Immunodeficient mice (NOG mice) were intraperitoneally transplanted with the cervical cancer cell line SiHa on day 4, and then divided into three groups. 1. HLA-edited FFluc-rejT+ NK cells (HLA-A24+) 2. HLA-edited FFluc-rejT+NK cells (HLA-A24-) 3.HLA editing FFluc-rejT On Day 0, groups 1 and 2 were assigned 2.5x10 6 rejT cells + 2.5 × 10 6 NK cells in group 3, and 2.5 x 10 6 Each group of rejT cells was administered intraperitoneally. The proliferation and persistence of rejT cells in the body were monitored by IVIS and compared. (result) The results are shown in Figure 5. In Group 3, where NK cells were not administered on Day 7, fluorescently labeled HPV-rejT efficiently proliferated and persisted in vivo, whereas in Group 2, it was eliminated by NK cells that did not have HLA-A24, making rejT undetectable on Day 7. On the other hand, in Group 1, fluorescently labeled HPV-rejT was not eliminated by NK cells that did have HLA-A24, and it was confirmed that it efficiently proliferated and persisted in vivo (left panel). When the persistence of fluorescently labeled HPV-rejT in mice on Day 7 was confirmed by signal, Group 1 had a significantly higher signal than Group 2, while there was no significant difference from Group 3 (right panel).

Claims

1. Cytotoxic T cells derived from human T cell-derived iPS cells express HLA-restricted class I of CTL antigen epitopes and HLA class I of HLA-E, with other HLA class I molecules being knocked out, wherein the HLA-restricted class I molecule of CTL antigen epitopes is HLA-A24 or HLA-A02.

2. Cytotoxic T cells derived from human T cell-derived iPS cells as described in claim 1, in which the HLA-restricted class I of the CTL antigen epitope and the HLA class I of HLA-E are exogenous, and all endogenous HLA class I has been knocked out.

3. A method for producing cytotoxic T cells derived from human T cell-derived iPS cells that express two types of HLA class I, HLA-restricted class I of the CTL antigen epitope and HLA-E, comprising the steps of: knocking out all HLA class I molecules of human T cell-derived iPS cells; introducing genes for HLA-restricted HLA class I and HLA-E of the CTL antigen epitope into the T-iPS cells in which all HLA class I molecules have been knocked out; and redifferentiating the gene-introduced T-iPS cells into CD8 single-positive T cells. The method for producing cytotoxic T cells derived from human T cell-derived iPS cells expresses two types of HLA class I, HLA-restricted class I of the CTL antigen epitope and HLA-E, comprising the steps of: knocking out all HLA class I molecules of human T cell-derived iPS cells;

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